本論文目的是欲探討鈣離子訊息於軟性基材(膠原蛋白凝膠)引發上皮細胞凋亡所扮演的角色。總共有三部份的研究包含於本論文中: 第一部份欲確認軟性基材引發細胞凋亡具有特定細胞類型的專一性；第二部份欲確認鈣離子恆定失調為軟性基材引發上皮細胞凋亡的主要原因；第三部份欲確認軟性基材引發細胞內鈣離子上升的主要原因是藉由活化STIM1分子而引發鈣離子內流所造成。
本論文第一部分的研究結果中顯示，膠原蛋白凝膠而非膠原蛋白凝膠鍍膜的培養皿會引發細胞凋亡，特別是在上皮細胞。軟性基材引發細胞凋亡具有上皮細胞專一性，但在間質細胞或癌症細胞則否。為了證實上述發現，利用年紀效應增加膠原蛋白纖維交聯程度的特性或利用超音波震盪以導致物理性破壞膠原蛋白纖維交聯程度來控制所製造之膠原蛋白凝膠的軟硬度，此結果顯示軟性基材引發上皮細胞凋亡可以被藉由增加基材的硬度所逆轉。
本論文的第二部分欲檢視軟性基材引發上皮細胞凋亡的機制為何？當LLC-PK1上皮細胞培養於軟性基材上時，可以發現caspase-12和caspase-3之前驅蛋白質的表現量減少，而且μ-calpain和caspase-3有發生活化現象。軟性基材同時也導致alpha-spectrin蛋白質被裂解，進而導致肌動蛋白細胞骨架瓦解。相對的，當上皮細胞培養於軟性基材上時，cytochrome c於粒腺體內的分布ヽ粒腺體膜電位ヽcaspase-9的活性以及caspase-8和caspase-9之前驅蛋白質的表現量並無顯著的改變，這表示內質網壓力而非死亡接受器或粒腺體所引發的細胞凋亡途徑可能牽涉於軟性基材引發之細胞凋亡。內質網壓力可能是因為內質網之鈣離子恆定機制失調或是過多的變異構型蛋白質分子堆積於內質網內所產生。軟性基材會導致細胞質內鈣離子濃度上升ヽ鈣離子於內質網過度累積及能容性鈣離子流(CCE)的提升。鈣離子螯合劑或鈣離子通道抑制劑可以藉由抑制μ-calpain的活性來部分降低軟性基材所引發的細胞凋亡。相反的，HeLa癌細胞不論是培養於膠原蛋白凝膠或是膠原蛋白凝膠鍍膜的培養皿上時，並不會發現有類似發生於LLC-PK1上皮細胞的眾多改變事件。因此，軟性基材所引發LLC-PK1上皮細胞凋亡主要是藉由擾亂細胞內鈣離子的恆定所造成。
最後，人類正常的子宮頸上皮細胞和子宮頸癌細胞被選擇用來從事對照研究，以比較細胞之鈣離子調控對於不同軟硬度之基材的反應。此部份的研究發現，軟性基材會藉由刺激並改變STIM1蛋白質分子(一個調控SOCC離子通道活性的關鍵調節分子)於細胞內位置的重新分布來提升正常子宮頸上皮細胞之能容性鈣離子流由細胞外通過細胞膜而進入細胞內。鈣離子恆定的瓦解會藉由提升細胞質內鈣離子濃度來活化μ-calpain，進而導致alpha-spectrin蛋白質的裂解ヽ肌動蛋白細胞骨架崩解與細胞凋亡。利用細胞外鈣離子螯合劑EGTA或STIM1小片段RNA技術來阻止細胞外鈣離子內流，幾乎可以完全抑制軟性基材所引發μ-calpain的活化，以及可以顯著地抑制軟性基材所引發正常的子宮頸上皮細胞凋亡。μ-calpain的抑制劑PD150606及PD151746，可以抑制軟性基材所引發細胞凋亡，並呈現出劑量依賴性的作用模式。利用μ-calpain小片段RNA技術降解細胞內之μ-calpain的表現量，同樣的也可以抑制軟性基材所引發細胞凋亡。相對的，子宮頸癌細胞培養於不同軟硬度之基材上，並不會影響細胞生長情況或細胞內鈣離子的恆定。因此，軟性基材對於活化不同種類細胞之STI1M分子所媒介之細胞外鈣離子內流能力的差異與軟性基材所引發之細胞凋亡密切相關。總結以上的研究發現，本論文對於鈣離子訊息在軟性基材引發上皮細胞凋亡的重要性提供了一個新的觀點。

The aim of this thesis is to study the role of Ca2+ signal in soft substrate (collagen gel)-induced epithelial cell apoptosis. Three parts of studies are included: (i) to identify soft substrate-induced apoptosis is cell-type specific; (ii) to identify the disturbance of Ca2+ homeostasis is the major cause of soft substrate-induced epithelial cell apoptosis; (iii) to identify STIM1-induced Ca2+ influx is the major cause of soft substrate-upregulated Ca2+ elevation.
The first part of this thesis showed that collagen gel, but not collagen gel-coating, induced apoptosis exclusively in epithelial cell lines. Soft substrate-induced cell apoptosis is specific in epithelial but not in mesenchymal or tumor cell lines. To confirm this, collagen gels with different rigidities due to cross-linking or physical disruption of collagen fibrils caused by sonication were used. In addition, soft substrate-induced epithelial apoptosis could be reversed by the increased substratum rigidity.
The second part of this thesis was to examine the underlying mechanisms of soft substrate-induced apoptosis in epithelial cells. Decrease of the protein levels of procaspase-12 and procaspase-3 when LLC-PK1 epithelial cells cultured on soft substrate. μ-calpain and caspase-3 were activated on soft substrate. Soft substrate also induced the cleavage of alpha-spectrin that resulted in the disorganization of actin cytoskeleton. In contrast, there was no significant change in cytochrome c revelation, mitochondrial membrane potential, caspase-9 activity and the protein levels of procaspase-8 and procaspase-9. This indicates that ER stress mediated- but not death receptor mediated- or mitochondria-mediated apoptotic pathway might involved in soft substrate-induced apoptosis. ER stress maybe caused by alternations in Ca2+ homeostasis and/or accumulation of misfolded proteins in the ER. Soft substrate caused elevated cytosolic Ca2+, Ca2+ overload in ER and upregulation of capacitative calcium entry. Ca2+ chelator or channel blocker partially rescued the soft substrate-induced apoptosis by inhibiting μ-calpain activation. In contrast, for HeLa tumor cells cultured either on collagen gel or on gel-coated dish, there was no significant change in above events. Thus, soft substrate induces apoptosis in LLC-PK1 cells by the disturbance of Ca2+ homeostasis.
Finally, normal cervical epithelial cells and cervical cancer cells were used as a pair to study cell-type specific Ca2+ regulation in response to soft substrate. Soft substrate upregulated the store-operated Ca2+ entry (SOCE) across the plasma membrane of normal cervical cells, which correlated with cellular redistribution of stromal interacting molecule 1 (STIM1), a key regulator of store-operated Ca2+ channel (SOCC). The disruption of Ca2+ homeostasis activated μ-calpain activity through rose intracellular Ca2+, resulting in alpha-spectrin cleavage, actin disorganization and subsequent apoptosis. Blockade of Ca2+ influx by EGTA or STIM1 siRNA almost abolished soft substrate-induced μ-calpain activation and significantly inhibited soft substrate-induced apoptosis. Calpain inhibitors, PD150606 and PD151746, dose-dependently inhibited soft substrate-induced apoptosis. Knockdown of μ-calpain by siRNA reduced soft substrate-induced apoptosis. In contrast, culture of cervical cancer cells on various substrates did not affect cell growth or disturb Ca2+ homeostasis. Thus differential activation of STIM1-mediated Ca2+ influx is associated with soft substrate-induced epithelial cell apoptosis. In conclusion, this thesis provides a new insight into the important role of Ca2+ signal in soft substrate-induced epithelial cell apoptosis.

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